谷氨酰胺抑制内质网应激反应改善大鼠创伤性颅脑损伤后神经功能

Glutamine supplementation provides neuroprotection by inhibiting endoplasmic reticulum stress response after traumatic brain injury in rats

目的 探讨谷氨酰胺（Gln）对大鼠创伤性颅脑损伤（TBI）后神经功能损伤评分、脑水肿、神经细胞凋亡和内质网应激反应的影响。方法 采用改良Feeney法建立大鼠TBI模型,将80只Sprague-Dawley（SD）大鼠按随机数字表法分为假手术组（Sham组），TBI组，TBI＋Gln处理组（TBI＋Gln组）和TBI＋Gln＋内质网应激诱导剂 2-脱氧葡萄糖组（TBI＋Glu＋2-DG组）。分别于伤后1、3、7、14 d进行神经行为学评分（mNSS）；干湿比重法检测损伤区脑皮质脑水含量；TUNEL染色检测神经细胞凋亡；免疫荧光、Westerrn blot方法测定凋亡相关因子［半胱氨酸蛋白酶（caspase）-12、caspase-3、Bcl-2］及内质网应激相关因子［肌醇需求酶1（IRE-1）、增强子结合蛋白同源蛋白（CHOP）］蛋白的表达。结果 与Sham组比较，其余3组脑水含量和mNSS损伤评分明显升高，脑组织凋亡相关因子（caspase-12、caspase-3、Bcl-2）和内质网应激相关因子（IRE-1、CHOP）蛋白表达上调（P均=0.00）；与TBI组比较，TBI＋Gln组脑水含量明显下降［3 d：（81.39±0.59）%比（83.54±0.52）%，P=0.04; 7 d：（74.86±0.38）%比（77.32±0.66）%,P=0.03］，治疗后7 d神经功能改善（8.63±0.22比10.37±0.29,P=0.03），凋亡因子caspase-12、caspase-3蛋白表达减少（P均=0.01），抗凋亡因子Bcl-2 表达增多（P=0.02），同时内质网应激相关因子IRE-1和CHOP蛋白表达减少（P均=0.00）。与TBI＋Gln组比较，TBI＋Glu＋2-DG组内质网应激相关因子表达增加，脑水含量增高，神经功能损伤加重（P＜0.05）。结论 谷氨酰胺处理能减轻伤后脑水肿，抑制神经细胞凋亡，改善TBI后神经功能行为，具有神经保护作用。其机制可能是通过抑制TBI后内质网应激反应来实现。

Objective To investigate the effects of glutamine （Gln） supplementation on neurologica severity score, brain edema, neuron apoptosis, and endoplasmic reticulum stress （ERS） response after traumatic brain injury （TBI） in rats. Methods TBI rat models were established using modified Feeneys method. Eighty Sprague-Dawley rats were divided into 4 groups with a random number table： sham operation group （Sham group）, TBI group, Gln supplementation group （TBI＋Gln group） and ERS inducer 2-deoxy-D-glucose group （TBI＋Gln＋2-DG group）. We measured the rats neurobehavioral outcomes by modified neurologic severity score （mNSS） on day 1, 3, 7 and 14 after TBI. Neuron apoptosis was detected using TUNEL staining. Brain water content was measured with wet-dry weight method.The apoptosis-related protein （caspase-12, caspase-3, and Bcl-2） and ERS-related cytokines [inositol-requiring enzyme 1（IRE-1）, C/EBP homologous protein （CHOP）] expressions in TBI cerebral cortex were determined by immunohistochemistry staining and Western blot. Results Compared with the Sham group, the levels of brain edema, mNSS, apoptosis-related protein （caspase-12, caspase-3, Bcl-2） and ERS-related proteins （IRE-1, CHOP） were significantly increased in the other three groups（all P=0.00）. Compared with the TBl group, the TBI＋Gln group showed significant lower brain water content[3 d：（81.39±0.59）% vs.（83.54±0.52）%,P=0.04; 7 d：（74.86±0.38）% vs. （77.32±0.66）%,P=0.03], improved mNSS（8.63±0.22 vs. 10.37±0.29,P=0.03）, suppressed expressions of apoptosis- and ERS-related proteins （caspase-12, caspase-3, IRE-1, and CHOP）（P=0.01, P〈0.01）, and increased expression of anti-apoptotic protein Bcl-2 （P=0.02）. Compared with the TBI＋Gln group, the expression of ERS-related factors （IRE-1 and CHOP）, brain edema level, and neurological severity were increased in the TBI＋Glu＋2-DG group. Conclusion Glutamine supplementation may have neuroprotection function, demonstrated as reducing brain edema and neuron apoptosis, and improving neurobehaviroal outcomes after TBI, possibly mediated by inhibiting TBI-induced ERS response.